1. Field of the Invention
This invention is related to the field of nucleic acid chemistry. Specifically, it is related to methods of amplifying nucleic acid sequences. The invention facilitates the amplification of nucleic acids under conditions of high fidelity. The invention may be used for a variety of industrial, medical and forensical purposes.
2. Description of the Invention
The polymerase chain reaction (PCR) is a well known in vitro method for the amplification of nucleic acid sequences (U.S. Pat. Nos. 4,683,202, 4,684,195, 4,965,188). The reaction uses two sequence specific oligonucleotide primers that hybridize to the opposite strands of the denatured target nucleic acid sequence. A heat-stable DNA polymerase catalyzes the elongation of the primers by incorporating desoxynucleotide monophosphates in the new strand.
The specificity of amplification depends on the specificity of primer hybridization. Under the elevated temperatures used in a typical PCR, the primers hybridize only to the target sequence. Under less stringent conditions, the primers may bind non-specifically to other nucleic acid sequences and initiate the synthesis of unspecific extension products. Amplification of unspecific PCR products can compete with the amplification of the target DNA and can significantly decrease the efficiency of the amplification of the target sequence.
In the past, several methods have been developed to reduce the formation of unspecific PCR products. In one method, referred to as a “hot-start” protocol, at least one critical reagent is withheld from the reaction mixture until the temperature is raised sufficiently to provide the necessary hybridization specificity. In this manner, the reaction mixture does not support the primer extension reaction until the missing component is added.
Hot start methods can be carried out manually by opening the reaction tube after an initial high temperature incubation step and adding the missing reagent. However, manual hot-start methods increase the risk of contamination and are labor intensive. Alternatively, heat labile materials, such as wax, are used to separate reaction components (U.S. Pat. No. 5,411,876). A high temperature pre-reaction incubation melts the heat labile material, thereby allowing the reagents to mix. Another method describes the use of antibodies to inhibit the DNA polymerase activity (U.S. Pat. No. 5,338,671). The antibodies are incubated with the polymerase prior to the set up of the reaction mixture to allow the formation of the antibody-DNA polymerase complex. Antibody inhibition is inactivated by denaturation of the antibody at a high temperature pre-incubation step. Additionally, the formation of extension product can also be inhibited by the addition of reagents like short oligonucleotides aptameres which bind to the DNA polymerase in a heat-reversible manner, thereby inhibiting polymerase activity (Lin & Jayasena (1997) J. Mol. Biol. 271: 100-111). However, the production of antibodies and aptameres is expensive and their application in a polymerase chain reaction may require redesign of the amplification reaction.
Non-specific amplification can also be reduced by the use of a reversibly inactivated thermostable DNA polymerase which can be reactivated by incubation in the amplification reaction mixture at an elevated temperature. Non-specific amplification is reduced because the polymerase is inactive until the temperature of the mixture has been elevated to a temperature which insures specific primer hybridization (U.S. Pat. Nos. 5,773,258; 5,677,152).
Routinely, PCR is performed using the thermostable DNA polymerase from Thermus aquaticus (Taq DNA polymerase) which shows a 5′-3′ polymerase activity and a 5′-3′ polymerase-dependent exonuclease function. However, it does not possess a 3′-5′ exonuclease activity (Lawyer et al. (1989) J. Biol. Chem. 264: 6427-6437). The 3′-5′ exonuclease activity of DNA polymerases is referred to as “proofreading activity”. This proofreading activity removes mismatched bases from the 3′ end of a primer-template duplex. It may be advantageous as it leads to an increased fidelity of replication during the amplification. As Taq DNA polymerase is deficient in 3′-5′ exonuclease activity it does not remove mismatched primer ends. However, it is able to elongate these mismatched primers thereby leading to an incorporation of base errors during amplification. Several thermostable B-type DNA polymerases exhibit 3′-5′ exonuclease activity and are used in PCR for the amplification of DNA with high fidelity. E.g., well known in the art are the DNA polymerases derived from Pyrococcus furiosus (Pfu DNA polymerase, WO 92/09689), Pyrococcus woesei (Pwo DNA polymerase available from Roche Applied Science) and Thermococcus gorgonarius (Tgo DNA polymerase, WO 981590).
Thermostable DNA polymerases with proofreading activity are also used in PCR as mixtures of DNA polymerases, at least one polymerase exhibiting such a proofreading activity (U.S. Pat. No. 5,436,149). Recently, a thermostable 3′-5′ exonuclease was shown to act as a mismatch correcting enzyme if used in PCR as a mixture with a DNA polymerase (WO 01/23583).
A repetitive series of cycles involving template denaturation, primer annealing, and extension of the annealed primers by the polymerase results in exponential accumulation of a specific DNA fragment. The primer extension products synthesized in a given cycle can serve as a template in the next cycle, therefore the number of target DNA copies approximately doubles every cycle. Thus, even smallest amounts of contaminating DNA from a previous PCR amplifications can be amplified and lead to false positive results (carry-over contamination). Therefore, methods have been developed to avoid such a contamination. In PCR amplifications it is possible to substitute dUTP for dTTP to produce uracil-containing DNA (U-DNA). Treating subsequent PCR reaction mixtures with uracil-DNA glycosylase (UNG) prior to amplification contaminating nucleic acids are degraded and are not suitable for amplification. dUTP can be readily incorporated by pol I-type thermostable DNA polymerases but not by B-type polymerases (Slupphaug et al. (1993) Anal. Biochem. 211:164-169). Therefore, B-type DNA polymerases can not be used in PCR amplifications if high fidelity and UNG decontamination is required.